Product Description

The ISOLATE II RNA Mini Kit provides a simple, efficient column-based method for the isolation of total RNA from a wide variety of starting materials, without the need for hazardous reagents such as phenol.

By combining the stringency of guanidinium-thiocyanate lysis with the speed and ease-of-use of silica-membrane purification, the ISOLATE II RNA Mini Kit provides a fast method for the purification of high-quality total RNA from animal and plant cells and tissues as well as cultured cells, bacterial cells, yeast, biological fluids and cell-free samples.

The online product manual has protocols for purifying total RNA from cultured cells, tissues, yeast, bacteria, biological liquids, paraffin embedded tissue and RNAlater® treated samples. There is also a protocol for a convenient on-column DNase treatment, using RNase-free DNase I that is supplied with the kit, for applications that are sensitive to very small amounts of DNA.

Applications

RT-qPCR

End-point RT-PCR

Northern, dot and slot blotting

Array analysis

Poly A+ RNA selection

RNA-Seq

High quality RNA

RNA was isolated from HeLa cells using ISOLATE II RNA Mini Kit and analyzed by the Bioanalyzer 2100 (Agilent Technologies). The results illustrate A) The quality of RNA, which was found to be exceptional (RIN: ≥9.2) and B) highly reproducible across samples.

Superior performance in real-time applications

RNA was isolated in a 10-fold serial dilution (14,000, 1,400, 140, 14 and 1.4 cells), from mouse 3T3 cells (lanes 1-5 respectively), using ISOLATE II RNA Mini Kit (red traces) and an equivalent kit from Supplier Q (green traces). Subsequently, real-time reverse transcriptase reactions were performed using SensiFAST SYBR No-ROX One-Step Kit. The results illustrate the higher quality of the extraction process over several orders of magnitude, down to a few cells.

Shipping conditions

FAQs

The columns supplied with the ISOLATE II kits may appear similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.

Interruption of the DNA/RNA extraction process is possible after the sample lysis only. It is possible to homogenize and lyse the samples and to store them in the freezer until use for RNA extraction. RNA clean up with a column cannot be interrupted and we recommend to avoid delays during the column purification process. If a delay is unavoidable the columns should be stored on ice.

An increase of A230 values may be caused by different substances, like carbohydrates, peptides and phenol. A bad A230/A260 ratio in RNA samples is mostly due to a contamination with guanidinium thiocyanate which is present in several reagents used for RNA extraction, for instance in the lysis buffer. In contrast to a bad A280/A260 ratio it does not automatically reflect a bad RNA quality. Currently there is no consensus about a lower limit of this ratio and mostly a carry-over of guanidinium thiocyanate does not affect the reliability of downstream applications. Nevertheless, an extra washing step with RW2 would be helpful to avoid this problem. And it would be helpful to pipet the flow-through out of the collection tube, instead of pouring it off.

For the optimal recovery of large RNA/DNA fragments it is necessary to optimize the elution step. To increase the recovery rate it would be helpful to use a larger volume for elution, incubate the column with the elution buffer prior to centrifugation and use repeated elution steps. To avoid excessive dilution it may be helpful to reapply the eluted fraction again.

Several possibilities exist. If you are using our column based extraction kit like the ISOLATE II RNA Mini kit it would be necessary to decrease the sample amount of such samples or to increase the amount of the lysis buffer RLY to prevent clogging of the columns. Another possibility would be a pre-extraction with TRIsure (BIO-38032) and to clean up the RNA containing aqueous phase with the column based ISOLATE II RNA kits. You should follow the TRIsure protocol up to the phase separation step. Then mix the aqueous phase with a volume of ethanol and load it onto the column. From there you should proceed with the regular ISOLATE II RNA Mini kit protocol.

We recommend the ISOLATE II Biofluids RNA kit for the extraction of cell free RNA. The kit isolates all sizes of RNA and is compatible with very limited, or challenging sample sources containing low RNA content.

We recommend our ISOLATE II Biofluids RNA, miRNA and Plant miRNA kits. The miRNA kits are able to capture all small RNA species (<200 nt) from plant cells and tissues and to purify high quality large RNA (>200 nt) from the same sample in parallel. The ISOLATE II miRNA Kits have been developed to overcome the bias observed with small RNA isolation using phenol-based techniques, due to its proprietary small RNA separation and enrichment technology, as well as highly optimized chemistry. The Biofluids RNA kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA down to small RNAs such as miRNA and siRNA. The ISOLATE II Biofluids RNA Kit is compatible with very limited, or challenging sample sources containing low RNA content.

Agarose gel analysis of RNA samples can be both valuable and misleading. The pattern of bands on the gel can not only be indicative of the quality of the sample, but equally it can also only indicate that the gel tank, buffer or agarose is contaminated with RNase. A safer measure is to use a Bioanalyzer and look at the RIN value (RNA Integrity Number), which should be as close to 10 as possible, indicating that the RNA is not degraded. A spectrophotometer (ideally a microfluidic one) can also be used to determine the ratio of A260 to A280 for purity determination.

All these methods give an idea of the quality of total RNA which tends to be dominated with rRNA and tRNA. It is not uncommon for an agarose gel to show an abundance of 18S and 23S rRNA but on analysis for there to be little of the transcript of interest. Ideally RNA quality control should include an assessment of the presence of common transcripts (from reference genes, such as GAPDH) using RT-PCR or RT-qPCR.

If the yield of RNA is low, it is best to first check that all the solutions used and the equipment employed is largely free of RNase. Solutions should be prepared with the highest quality reagents available, preferably ones that have a certificate of analysis to show that they are free of DNase and RNase. If there is confidence that RNase contamination is at a minimum, then it may be worth ensuring that the sample is properly homogenised before lysis and to check a sample of the lysed material under a light microscope to ensure that all the cells are disrupted.

RNA samples that have been contaminated with RNase (either during processing or those that have been sent from another lab) can be purified using the ISOLATE II RNA Micro Kit.

Reviews

"I recently used the ISOLATE II RNA Mini kit. I had some cultured cells which ... "

Sharon Pok, Australian National University Medical School, Garran, Australia

"We have been utilizing ISOLATE II RNA Mini Kit from Bioline and have found ... "

Aparna Jayachandran, GMRF-UQ, Brisbane, Australia

""I find that the 260/280 and 260/230 ratios with this kit (ISOLATE II ... "

Newcastle University, UK

"I find that the 260/280 and 260/230 ratios with this kit (ISOLATE II RNA Mini Kit) are excellent and reproducible. I previously used the Qiagen RNeasy kit, but the Bioline kit is both cheaper and gives better quality RNA."

Newcastle University, UK

We have been utilizing ISOLATE II RNA Mini Kit from Bioline and have found that the RNA yield, concentrations and purity have been consistently good. From 40,000 liver cancer (mouse and human) cells we are able to get 200-600ng/ul yield of RNA.

Aparna Jayachandran, GMRF-UQ, Brisbane, Australia

I recently used the ISOLATE II RNA Mini kit. I had some cultured cells which are derived after FACS, which are not very many, to extract RNA from. Because I had a small starting quantity (<1E6 cells), I wasn’t expecting an extremely high yield. However, I attained a relatively good yield from that small amount of starting sample. What also impressed me was the 260/280 and 260/230 ratios. I have been having trouble getting a good 260/230 ratio lately, probably because of residual guanidine not being washed off properly. With the kit, both ratios were well above 2.0.

Sharon Pok, Australian National University Medical School, Garran, Australia

We have been using Bioline products (especially the Isolate II RNA mini Kit, cDNA synthesis Kit and Sensifast SBYR mix) for past 3 years, and we are very happy with the consistency of results that we obtained and also the good price they offered. We are happy to continue using these products.